Evaluation of the influence of the base material loaded with hydrous zirconium oxide gel on the retention behavior of disubstituted benzoic acids

Summary Silica gel loaded with hydrous zirconium oxide (Zr-gel-S) has been prepared and the influence of the base material on the retention behavior ofo-phthalic acid on the hydrous zirconium oxide was evaluated. The retention behavior of the acid on the Zr-gel-S was compared with that on Zr-gel-5, the polymer-based Zr-gel. Comparable retention behavior,i.e. maximum retention in the vicinity of pH 6, which has been regarded as the equal adsorbic point, was observed for both types of Zr-gel, although there was a large difference in the retention times. The difference in retention on the two types of Zr-gel could be ascribed to nature of the base material. The results in this experiments showed, however, that the specific behavior at pH 6 was an essential characteristic of hydrous zirconium oxide and was not related to the influence of the base materials.     

Liquid chromatography of isomers of aromatic acids on microbore column packed with anion-exchange resin

Summary A low-capacity, anion-exchange, porous-polymer resin was used as the packing material in a microbore column. Using a phosphate buffer containing various organic modifiers, retention behavior of substituted benzoic acid isomers with amino, chloro, methyl, nitro and carboxylic groups was investigated. The effect of ethanol, acetonitrile and sodium dodecylsulfate used as organic modifiers on the retention behavior at various eluent pH values was studies and optimum separations of the positional isomers reported.     

Estimation of the pK a of the amine groups of benzhydrylamine resin and its lysyl derivative by measurement of swelling

Summary For better evaluation of the potential of benzhydrylamine resin (BHAR), used as a solid support for peptide synthesis, as a novel anion-exchange resin, the pK _a of its amine group was estimated by microscopic measurement of the sizes of the dry and swollen beads. Using the volume of the bead occupied by the solvent (as a percentage) as the swelling parameter, a plot of the degree of swelling of BHAR loaded with 2.4 mmol g^–1 amine groups against the pH of the medium produced a decreasing sigmoidal-type curve with increasing pH. By considering the point of inflection of the curve, a pK _a value of approximately 7.5 was estimated for the amine group of the BHAR. The same approach was also applied to the lysyl derivative of the BHAR (Lys-BHAR) and pK _a values around 6.5 and 10.0 were obtained for the and amine groups, respectively.     

Simultaneous HPLC Determination of Five Flavonoids in Flos Inulae

A simple and accurate reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for simultaneous determination of five flavonoids, spinacetin, quercetin, luteolin, 6-methoxyluteolin, and isorhamnetin, in an extract of the flowers of Inula britannica L., an important Traditional Chinese Medicine (TCM). Samples were extracted with 80% ethanol. Optimum separation and detection were achieved on an ODS-3 column with a methanol–acetonitrile gradient containing 0.49% (v/v) citric acid as mobile phase. The flow rate was 1.0 mL min⁻¹ and detection was at 360 nm. All calibration plots revealed linearity was good (r ² = 0.999) within the concentration ranges tested. Repeatability was evaluated by performing intra-day and inter-day assays; relative standards deviations (RSD) were less than 2.8%. Recovery of the five flavonoids was between 91.5 and 103.6%, with RSD less than 6.5%. The method was successfully used for analysis of seven samples of Flos Inulae from different parts of China and was found to be simple and efficient.     

Determination of the five major opium alkaloids by reversed-phase high-performance liquid chromatography on a base-deactivated stationary phase

Summary A reversed phase HPLC method for the separation of the five major alkaloids fromPapaver somniferum L., morphine, codeine, thebaine, papaverine and noscapine, has been developed and validated. By use of a basedeactivated silica-based stationary phase excellent peak shape was achieved for each substance. The five alkaloids were quantified by internal standardization within 20 min and with good precision. The method is applicable to opium and to poppy straw.     

Identification of the major compounds in extracts ofVerbena officinalis L. (Verbenaceae) by HPLC with post-column derivatization

Summary A qualitative reversed-phase HPLC method has been developed for the analysis of 50% MeOH extracts ofVerbena officinalis L. (Verbenaceae) leaves. The method enables separation of the main constituents: iridoids, flavonoids and phenolic acid derivatives. Simultaneous detection at different wavelengths, measurement of the UV spectrum of each separated compound during elution and co-injection of reference substances facilitated easy and rapid identification of verbenalin, hastatoside and verbascoside. As some of these compounds, mainly flavonoids, have closely related structures, however, characterization by derivatization with reagents inducing a shift of UV absorption maxima was required. This furnished additional structural information. The reagents were adapted for compatibility with the solvent system used for the chromatographic separation.     

Determination of Homoisoflavonoids in Ophiopogon japonicus by RP-HPLC

The homoisoflavonoid content of 14 samples of Ophiopogon japonicus collected from different parts of China have been investigated. Three principal homoisoflavonoids, methylophiopogonanone A, methylophiopogonanone B, and 6-aldehydo-isoophiopogonanone A, were analyzed simultaneously by reversed-phase high-performance liquid chromatography with a binary mixture of acetonitrile and 0.3% aqueous acetic acid as mobile phase. The recovery of the method was 98.5–102.6% and good linearity (r > 0.9990) was obtained for all the homoisoflavonoids over a relatively wide concentration range. The results indicated that the homoisoflavonoid content of Ophiopogon japonicus varied significantly from species to species, and from locality to locality. The quality and identity of Ophiopogon japonicus from the herb market can be determined from the homoisoflavonoid content.     

Rapid Determination of Aristolochic Acids I and II in Some Medicinal Plants by High-Performance Liquid Chromatography

Aristolochic acids (AA) are toxic components of Aristolochia plants which result in diseases of the kidney such as urothelial cancer. It is, therefore, essential to monitor the amount of aristolochic acid in herbal medicines. In this study a reversed-phase high-performance liquid-chromatographic (HPLC) method has been developed for rapid determination of aristolochic acids I and II. Baseline separation was achieved within five minutes by use of an ODS C₁₈ column with methanol–water, 60:40, as mobile phase. Two kinds of aristolochic acid were successfully determined in 31 herbal samples of Aristolochia fangchi Wu and Caulis Aristolochiae Manshuriensis. The results indicated that in most samples the aristolochic acid I content is much higher than that of aristolochic acid II. The two kinds of aristolochic acid were not detected in Aristolochia fangchi from the Guangdong region, so Aristolochia fangchi from this region is recommended for use in herbal remedies.     

Determination of naturally occurring hydroxynaphthoquinone polymers by size-exclusion chromatography

Summary Polymerization of alkannin, shikonin, and their derivatives, potent pharmaceutical substances, crucially affects their use in pharmaceuticals, cosmetics, and as food colorants, because it leads to loss of their antimicrobial activity, reduction of the lustre of their red coloration, and a decrease in their solubility. In this study size-exclusion chromatography (SEC) has been used for the first time for qualitative and quantitative analysis of monomeric and polymeric hydroxynaphthoquinone alkannin and shikonin derivatives. The purity and degree of polymerization has been determined to evaluate severalAlkanna tinctoria root samples from different geographical sources, and commercial samples of alkannin and shikonin, as pharmaceutical raw materials. Conditions for extraction of hydroxynaphthoquinones fromAlkanna tinctoria roots with olive oil were optimized in terms of polimerization, aiming to improve the biological activity of the final pharmaceutical product, Helixderm.     

Determination of N-Acetylcysteine and Thioglycolic Acid in Human Urine

A method for the determination of total N-acetylcysteine and thioglycolic acid in human urine is described. Because these compounds are mainly excreted as disulfides, they are first reduced to the free thiols by treatment with tris(2-carboxyethyl)phosphine hydrochloride and then derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. Separation and quantitation of the 2-S-quinolinium derivatives of the thiols were achieved by reversed-phase ion-pair liquid chromatography with UV-detection at 355 nm. Because the method enables simultaneous determination of other endogenous urinary thiols, e.g. cysteine and cysteinylglycine, amounts of these compounds in urine were also studied. Detector responses were linear over the range covering most practical situations, with correlation coefficients for all four analytes better than 0.999. Recovery and imprecision (as RSD) were within 99.77–102.17 and 0.01–7.79%, respectively. The lower limit of detection was 0.25 μmol L⁻¹ urine for thioglycolic acid and N-acetylcysteine, and 0.12 μmol L⁻¹ urine for cysteine and cysteinylglycine. The method was used for analysis of urine samples from 29 healthy individuals to establish reference values for the thiols, normalized to creatinine. 3-Mercaptolactic acid, 2-mercaptopropionic acid, and mercaptoethanol were not present in the urine analyzed.     

Simultaneous Determination of Six Major Constituents in the Stems of Kadsura heteroclita by LC-DAD

The stem of Kadsura heteroclita is a traditional Chinese medicine with a variety of biological activities. For efficient monitoring of the quality of the herb, a simple, rapid, and accurate HPLC method has been developed for simultaneous determination of five cyclolanostane triterpenoid compounds (schisanlactone E, nigranoic acid, schisandronic acid, and heteroclitalactones A and D) and one tetrahydrofuran lignan (d-epigalbacin) in the stems of K. heteroclita. These six compounds were separated on a C₁₈ column by elution with a mobile-phase gradient prepared from acetonitrile and 0.05% (v/v) aqueous phosphoric acid. The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 210 nm. The recovery of the method was in the range 95.03–102.19% and a good linear relationship (r ² > 0.9999) was obtained between response and compound concentration over a relatively wide range. The method was successfully applied to analysis of the herb collected at different stages of growth.     

HPLC separation of opium alkaloids on porous and non-porous stationary phases

Summary A new reversed-phase (RP) HPLC method has been developed and validated for the separation of the main opium alkaloids morphine, codeine, thebaine, papaverine and noscapine on a non-porous (micropellicular) stationary phase. On this phase quantification of the compounds by internal standardization with brucine was achieved extremely rapidly, in ca 1.5 min, only. Thus, the analysis time for the opium alkaloids was approximately one tenth of that on porous stationary phases. Different opium samples were investigated using non-porous and porous packings. The correlation between the results was excellent. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

High-Performance Liquid Chromatographic Method for Determination and Pharmacokinetic Study of Chlorogenic Acid in the Plasma of Rats After Administration of the Chinese Medicinal Preparation Luying Decoction

A simple and sensitive high-performance liquid chromatographic method has been developed for determination of chlorogenic acid in rat plasma. Chlorogenic acid was extracted from plasma samples with methanol. HPLC analysis of the extracts was performed on a C₁₈ column (250 mm × 4.6 mm i.d., 5 µm particles). The mobile phase was acetonitrile −1% formic acid (9:91, v/v). The calibration plot was linear over the range 0.0420–2.10 µg mL⁻¹ and the lower limit of quantification was 0.0420 µg mL⁻¹. The method was reproducible and reliable with intra-day precision better than 8.2%, inter-day precision better than 9.1%, accuracy within ±8.3%, and mean extraction recovery above 84.4%. The validated method was successfully applied to pharmacokinetic studies of chlorogenic acid in rat plasma after administration of Luying decoction.     

Liquid Chromatography for Separation and Quantitative Determination of Adrenergic Amines and Flavonoids from Poncirus trifoliatus Raf. Fruits at Different Stages of Growth

The fruits of Poncirus trifoliatus Raf. (Rutaceae) have been used against allergic diseases for generations and still occupy an important place in traditional oriental medicine. They have also been used to treat gastric and duodenal ulcers. Chemical analysis of extracts of Poncirus trifoliatus Raf. fruit at different stages of maturation revealed variations in the concentrations of flavonoids present. Fourteen flavonoids (neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, hesperetin, sinensetin, nobiletin, heptamethoxyflavone, 5-O-demethylnobiletin, and tangeretin) and five amines (synephrine, octopamine, N-methyltyramine, hordenine, and tyramine) in extracts from the fruit of Poncirus trifoliatus Raf. were analyzed by HPLC with a C₁₈ reversed-phase column. The concentrations of the four flavonoids naringin, poncirin, narirutin, and neoponcirin was maximum during the first stage of growth and gradually decreased until the fruits were fully developed. The paper also discusses the anatomical variations observed at different stages of development of the fruit.     

Determination of the curcuminoid pigments in turmeric (Curcuma domestica Val) by reversed-phase high-performance liquid chromatography

Summary A rapid, simple and reproducible reversed-phase high-performance liquid chromatographic method was developed for the quantitative determination of curcumin, demethoxycurcumin and bis-demethoxycurcumin in ethanolic extracts of turmeric. The pigments were separated on a styrene-divinylbenzene copolymer column (Hamilton PRP-1), using an acetonitrile-water (5545, v/v) mobile phase. The pigments were monitored with a diode-array detector at 425 nm. The limit of detection was 10.2 ng curcumin, 11.1 ng demethoxycurcumin and 6.2 ng bis-demethoxycurcumin. Comparison of HPLC and spectrophotometric results for the determination of the total curcuminoid content for a number of turmeric samples, reveal that the spectrophotometric method invariably yielded higher results, indicating an overestimation of curcuminoids.     

Mechanistic study of the sorption properties of OASIS® HLB and its use in solid-phase extraction

Summary The solvation parameter model is used to characterize the sorption properties of the porous polymer Oasis^? HLB for solid-phase extraction with water and water-methanol mixtures as a sample solvent. Increasing solute size and electron lone pair interactions favor retention from water. Oasis^? HLB is not competitive with water for dipole-type and hydrogen-bond interactions, which result in lower analyte retention. The selectivity of Oasis^? HLB is different to porous graphic carbon (Hypercarb^?), a conventional poly (styrene-divinylbenzene) porous polymer sorbent (PLRP-S 100) and two silica-based, octadecylsiloxane-bonded sorbents with a high and a low carbon loading, respectively. Because of selectivity differences no single sorbent is ideal for the extraction of analytes possessing a wide range of polar interactions. Oasis^? HLB is preferred for the extraction of low molecular mass and polar compounds, PLRP-S 100 for the extraction of higher molecular mass compounds of moderate polarity, and the silica-based octadecylsiloxane sorbent with a high carbon loading is the best compromise for the extraction of compounds that cover a wide polarity range. For methanol-water mixtures as a sample solvent PLRP-S 100 is the best general choice with Oasis^? HLB preferred for the extraction of strong hydrogen-bond acids. Hypercarb^? is shown to have favorable retention properties for solid-phase extraction with the except for its low surface area.     

Determination ofm-xylylenediamine in the official EU food simulant olive oil

Summary EU Directive 90/128/EEC prescribes a specific migration limit (SML) of 0.05 mg kg⁻¹ for the aliphatic diaminem-xylylenediamine (XDA) into food or food simulants, but there is no generally accepted method of analysis available for testing compliance with this restriction.m-XDA has been determined in olive oil (EU food simulant for fatty foods) by high-performance liquid chromatography (HPLC) with fluorescence detection, after extraction into an aqueous phase and derivatization with fluorescamine. The method is appropriate for the quantitative determination ofm-XDA at a minimum level of 0.02 mg kg⁻¹ in this food simulant. The method should also be applicable to other fatty-food simulants (sunflower oil and HB 307).     

Determination of monensins A and B in the fermentation broth ofStreptomyces cinnamonensis by high performance liquid chromatography

Summary High performance liquid chromatography on an octadecyl silica column has been used to determine both the monensin A: monensin B ratio and by the method of standard addition, the concentration of both in the fermentation broth ofStreptomyces cinnamonensis. Refractive index detection was preferred to ultraviolet owing to the presence of UV-absorbing components which could not be completely separated from the substances of interest. A linear relationship was obtained from the calibration data. The coefficients of variation for the estimation both of the ratio and the concentrations of the compounds were better then 5%. The estimated limit of detection for both substances was about 1 g/ml. The results obtained from the determination of the ratios of monensins were compared with those obtained by chemical ionization mass spectrometry. Chromatographic separation of monensins on the silica gel column is also described.     

RP-LC Determination of Rutin in Amaranthus spinosus Linn. Whole Plant Powder

A simple, reproducible and efficient reverse phase high performance liquid chromatographic method has been developed for the determination of rutin in whole plant powder of Amaranthus spinosus Linn. The analysis was done with Waters Cosmosil C₁₈ column (150 × 4.6 mm i.d., 5 μm particle size) as stationary phase at a wavelength of 361 nm for detection and determination. The proposed HPLC method was validated for linearity, accuracy, precision and limit of quantitation.     

Ligand-exchange chromatography of amino acid enantiomers and its application to the biotransformation of DL-aspartic acid byPseudomonas dacunhae

Summary The separation of the D and L enantiomers of eighteen essential α amino acids has been investigated by ligand-exchange chromatography (LEC). The effect of column temperature on the retention times and resolution of individual amino acid enantiomers has been studied by varying the temperature from 25 to 50 °C for a mobile phase containing Cu²⁺ ions. By use of a temperature of 50 °C and Zn²⁺ in the mobile phase, eight of the eighteen amino acid enantiomers can be resolved sufficiently well for practical application. Only phenylalamine, tyrosine, and tryptophan can be separated by use of Ni²⁺ as complexation metal at 50 °C. LEC has been used to monitor the decarboxylation of racemic DL-aspartic acid byPseudomonas dacunhae. Analysis of DL amino acid enantiomers in different media was performed at column temperatures of 30 and 50°C by addition of 0.125 mM Cu²⁺ to the aqueous mobile phase. It was found that the analytical performance is most dependent on the identity of the metal used for complexation; the concentration of the metal was of secondary importance and the column temperature less important still.